Project Summary Electrically excitable cells are spread throughout the human body, playing crucial roles in the development and function of all organs, and are particularly fundamental to the genesis and normal activity of the nervous system. Ligand-gated ion channels (LGICs) are multimeric integral membrane proteins that reside on the cellular membranes of excitable cells and transduce the binding of small molecule ligands ? so called chemical transmitters ? into the opening of an ion conducting channel across the cell membrane, thereby transducing a gradient of a chemical into an electrical signal that, in turn, is propagated to adjacent cells. Because of the central roles that LGICs play in the activities of excitable cells, and because excitable cells underpin the function of many organs and of the nervous system, LGICs are the targets of a large number of therapeutic drugs, including sedatives, anticonvulsive agents, and anti nausea medications. Furthermore, multiple diseases and disorders are associated with the dysfunction of LGICs. Pentameric Cys-loop receptors and ATP-gated P2X receptors are two of the most important and widespread classes of LGICs, are widely expressed throughout the human body and are long standing targets of therapeutic agents. Moreover, these two classes of LGICs have been the subject of intensive biophysical and biochemical studies for decades. Despite the significance and historical importance of LGICs in medicine and basic science, the study of their 3D structures has proven difficult. The research proposed in this application will apply new technologies and reagents to dramatically advance the structural study of two Cys-loop receptor subtypes ? the glycine and GABAA receptor ? and of trimeric, ATP-gated P2X receptors. Glycine and GABAA receptors are particularly important because they set the inhibitory `tone' of the peripheral and central nervous systems, respectively, and are the targets of a large number of highly efficacious therapeutic agents. Moreover, P2X receptors are emerging targets for the treatment of pain, inflammation and cough. Despite the prominence of glycine and GABAA receptors, as well as P2X receptors, in human biology and disease, we do not understand, in molecular detail, how the receptors are activated by agonists, inhibited by antagonists, and modulated by the action of small molecules binding at novel sites. Moreover, we do not understand how these LGICs transition from agonist-bound open states to agonist-bound desensitized or inactive states. Using cutting edge single particle cryo-electron microscopy methods and x-ray diffraction approaches, together with novel reagents and methods, we will define mechanisms for the activity of the glycine and GABAA receptors, as well as for the human P2X3 receptor. These advances, in turn, will provide a foundation for the development of novel therapeutic agents and for a deeper understanding of the relationships between atomic structure and molecular function in the Cys-loop and P2X receptor families.